EP2183379B1 - Enrichissement d'une séquence cible - Google Patents

Enrichissement d'une séquence cible Download PDF

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Publication number
EP2183379B1
EP2183379B1 EP20080794916 EP08794916A EP2183379B1 EP 2183379 B1 EP2183379 B1 EP 2183379B1 EP 20080794916 EP20080794916 EP 20080794916 EP 08794916 A EP08794916 A EP 08794916A EP 2183379 B1 EP2183379 B1 EP 2183379B1
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EP
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Prior art keywords
target
sequence
reference
temperature
method
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EP20080794916
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German (de)
English (en)
Other versions
EP2183379A4 (fr )
EP2183379A2 (fr )
Inventor
Gerassimos Makrigiorgos
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Dana-Farber Cancer Institute Inc
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Dana-Farber Cancer Institute Inc
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6858Allele-specific amplification

Claims (15)

  1. Méthode d'enrichissement d'une séquence cible dans un mélange de réaction, ladite méthode comprenant :
    a. soumettre un mélange de réaction dont on suspecte qu'il contient un duplex de séquence cible et un duplex de séquence de référence à une première température de dénaturation qui est au-dessus de la température de fusion (Tm) du duplex de séquence de référence et du duplex de séquence cible de manière à permettre la dénaturation dudit duplex de séquence cible et dudit duplex de séquence de référence, où ledit duplex de séquence cible comprend une ou plusieurs insertion, délétion ou altération et diffère par au moins un nucléotide dudit duplex de séquence de référence, et où ladite séquence cible est au moins à 50% homologue audit duplex de séquence de référence et est amplifiable par la même paire d'amorces que ledit duplex de séquence de référence, et où la séquence cible est moins prévalente dans le mélange de réaction que la séquence de référence
    b. réduire la température du mélange de réaction à une température d'hybridation de manière à permettre la formation de duplexes de brin cible/brin de référence et à prévenir la liaison de ladite paire d'amorces à la cible dénaturée et aux brins de référence dans le mélange de réaction, où la température hybridation est en dessous de la température critique (Tc) et au-dessus de la température de recuit d'amorce ;
    c. augmenter ledit mélange de réaction d'amplification à une température critique (Tc) qui est en dessous de la Tm dudit duplex de séquence de référence de manière à permettre la dénaturation préférentielle desdits duplexes de l'étape (b) pour former des brins cible et de référence dénaturés ;
    d. réduire la température du mélange de réaction à une température de recuit d'amorce de manière à permettre à ladite paire d'amorces de recuire auxdits brins cible et de référence ; et
    e. étendre ladite pair d'amorces de manière à enrichir ladite séquence cible relativement à ladite séquence de référence ;
    où la méthode est répétée pendant deux ou plusieurs cycles.
  2. Méthode selon la revendication 1, dans laquelle lesdites séquences cible et de référence sont d'abord amplifiées en soumettant le mélange de réaction à une PCR.
  3. Méthode selon la revendication 1, dans laquelle ladite séquence cible est un allèle mutant différant de la séquence de référence selon entre 1 et 10 nucléotides.
  4. Méthode selon l'une quelconque des revendications 1 à 3, dans laquelle :
    (i) lesdites séquences cible et de référence comprennent 25 à 500 bases, et/ou
    (ii) ladite Tc est 0,3°C à 5°C en dessous de la Tm de ladite séquence de référence, et/ou
    (iii) ladite Tc est en dessous de la Tm de la séquence cible et/ou
    (iv) ladite méthode est répétée entre 5 et 40 cycles et/ou
    (v) ladite méthode est répétée entre 10 et 30 cycles.
  5. Méthode selon l'une quelconque des revendications 1 à 4, comprenant en outre l'étape consistant à analyser ledit mélange de réaction avec la séquence cible enrichie,
    où ladite analyse utilise en option une ou plusieurs des méthodes sélectionnées dans le groupe consistant en : MALDI-TOF, Fusion HR, séquençage di-désoxy, séquençage à molécule unique, pyroséquençage, SSCP, RFLP, dHPLC, PCR numérique et PCR quantitative.
  6. Méthode selon la revendication 1, dans laquelle ladite Tc est appliquée entre 1 seconde et 5 minutes.
  7. Méthode selon l'une quelconque des revendications précédentes, dans laquelle ladite séquence cible est méthylée d'une manière différente de la séquence de référence, et avant la mise en oeuvre de la méthode selon la revendication 1 sur le mélange de réaction, le mélange de réaction étant traité en option avec du sodium bisulfite.
  8. Méthode selon l'une quelconque des revendications précédentes, dans laquelle ledit mélange de réaction contient un colorant de détection de l'acide nucléique, et est en option exécutée dans un dispositif PCR en temps réel.
  9. Méthode selon la revendication 1 exécutée sous des conditions de réaction en temps réel en utilisant un agent de détection d'acide nucléique.
  10. Méthode selon la revendication 1, dans laquelle ladite méthode est utilisée pour enrichir deux ou plusieurs séquences cibles différentes, et ladite méthode comprend en outre une ou plusieurs paires additionnelles d'amorces spécifiques auxdites séquences cibles.
  11. Méthode selon la revendication 1, dans laquelle ladite paire d'amorces possède une température de fusion qui est en dessous de la température appliquée à l'étape (b), en option au moins 5°C en dessous de la température dans l'étape (b).
  12. Méthode selon la revendication 1, dans laquelle le mélange de réaction comprend un acide nucléique modifié.
  13. Procédé d'enrichissement d'une séquence cible selon la revendication 1, comprenant en outre :
    répéter les étapes b) et c) plus d'une fois par cycle avant l'exécution de l'étape d).
  14. Procédé selon la revendication 13, dans laquelle la séquence cible est au moins à 70% homologue avec la séquence de référence, et de préférence au moins à 80% homologue à la séquence de référence.
  15. Support lisible par ordinateur comprenant des instructions de programme aptes à exécuter la méthode selon la revendication 1.
EP20080794916 2007-08-01 2008-07-31 Enrichissement d'une séquence cible Active EP2183379B1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US96283807 true 2007-08-01 2007-08-01
PCT/US2008/009248 WO2009017784A3 (fr) 2007-08-01 2008-07-31 Enrichissement d'une séquence cible

Publications (3)

Publication Number Publication Date
EP2183379A2 true EP2183379A2 (fr) 2010-05-12
EP2183379A4 true EP2183379A4 (fr) 2011-08-10
EP2183379B1 true EP2183379B1 (fr) 2015-04-15

Family

ID=40305136

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20080794916 Active EP2183379B1 (fr) 2007-08-01 2008-07-31 Enrichissement d'une séquence cible

Country Status (9)

Country Link
US (1) US8455190B2 (fr)
EP (1) EP2183379B1 (fr)
JP (1) JP5531367B2 (fr)
KR (1) KR101376359B1 (fr)
CN (1) CN101815789B (fr)
CA (1) CA2695264C (fr)
DK (1) DK2183379T3 (fr)
ES (1) ES2547053T3 (fr)
WO (1) WO2009017784A3 (fr)

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CA2830361A1 (fr) * 2011-03-31 2012-10-04 Dana-Farber Cancer Institute, Inc. Procedes et compositions pour permettre une cold-pcr multiplexe
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CN105283555A (zh) 2013-10-20 2016-01-27 特罗瓦基因公司 核酸序列的合成和富集
CA2922261A1 (fr) * 2013-10-20 2015-05-21 Trovagene, Inc. Synthese et enrichissement de sequences d'acides nucleiques
CN103602748A (zh) * 2013-11-28 2014-02-26 瑞希基因科技(北京)股份有限公司 一种结合荧光定量pcr技术的焦磷酸测序方法
WO2016007914A1 (fr) * 2014-07-10 2016-01-14 Fluoresentric, Inc. Technologie d'amplification d'adn
WO2017070339A1 (fr) * 2015-10-20 2017-04-27 Richardson Katherine Dispositif microfluidique pour l'enrichissement de modifications de séquence d'acide nucléique
WO2018111835A1 (fr) * 2016-12-12 2018-06-21 Dana-Farber Cancer Institute, Inc. Compositions et procédés pour le codage par code-barres moléculaire de molécules d'adn avant l'enrichissement des mutations et/ou la détection des mutations

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Also Published As

Publication number Publication date Type
ES2547053T3 (es) 2015-10-01 grant
EP2183379A4 (fr) 2011-08-10 application
DK2183379T3 (da) 2015-07-27 grant
KR101376359B1 (ko) 2014-03-27 grant
CN101815789A (zh) 2010-08-25 application
CA2695264A1 (fr) 2009-02-05 application
JP5531367B2 (ja) 2014-06-25 grant
EP2183379A2 (fr) 2010-05-12 application
WO2009017784A3 (fr) 2009-03-26 application
KR20100044870A (ko) 2010-04-30 application
US20100203532A1 (en) 2010-08-12 application
US8455190B2 (en) 2013-06-04 grant
WO2009017784A2 (fr) 2009-02-05 application
CA2695264C (fr) 2015-01-27 grant
JP2010535031A (ja) 2010-11-18 application
CN101815789B (zh) 2013-06-12 grant

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